Transcriptomic Responses to Polymyxin B and Analogues in Human Kidney Tubular Cells

Polymyxins are last-line antibiotics for the treatment of Gram-negative ‘superbugs’. However, nephrotoxicity can occur in up to 60% of patients administered intravenous polymyxins. The mechanisms underpinning nephrotoxicity remain unclear. To understand polymyxin-induced nephrotoxicity, human renal proximal tubule cells were treated for 24 h with 0.1 mM polymyxin B or two new analogues, FADDI-251 or FADDI-287. Transcriptomic analysis was performed, and differentially expressed genes (DEGs) were identified using ANOVA (FDR < 0.2). Cell viability following treatment with polymyxin B, FADDI-251 or FADDI-287 was 66.0 ± 5.33%, 89.3 ± 3.96% and 90.4 ± 1.18%, respectively. Transcriptomics identified 430, 193 and 150 DEGs with polymyxin B, FADDI-251 and FADDI-287, respectively. Genes involved with metallothioneins and Toll-like receptor pathways were significantly perturbed by all polymyxins. Only polymyxin B induced perturbations in signal transduction, including FGFR2 and MAPK signaling. SIGNOR network analysis showed all treatments affected essential regulators in the immune system, autophagy, cell cycle, oxidative stress and apoptosis. All polymyxins caused significant perturbations of metal homeostasis and TLR signaling, while polymyxin B caused the most dramatic perturbations of the transcriptome. This study reveals the impact of polymyxin structure modifications on transcriptomic responses in human renal tubular cells and provides important information for designing safer new-generation polymyxins.


Introduction
The polymyxins, namely polymyxin B and colistin (polymyxin E), have been used clinically since the late 1950s to treat infections caused by multidrug-resistant (MDR) Gramnegative bacteria, but were largely abandoned in the 1970s due to a high incidence of nephrotoxicity following intravenous administration [1,2]. However, the emergence of MDR Gram-negative bacteria resistant to virtually all currently available antibiotics has seen the reintroduction of the polymyxins into the clinic, in particular for treatment of Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacterales infections [3]. Given that the polymyxins retain substantial activity against many MDR Gram-negative bacteria, they are now often used as a last-line therapy to combat infections caused by these pathogens [4]. Nevertheless, nephrotoxicity remains the major dose-limiting factor for polymyxin use, 2 of 14 occurring in up to 60% of patients [4]. Unfortunately, there is uncertainty regarding the mechanism by which polymyxins induce nephrotoxicity. Polymyxins are known to be extensively reabsorbed by and accumulate within kidney tubular cells [5,6], with necrosis subsequently occurring in a dose-and time-dependent manner [7]. Both polymyxin B and colistin have been shown to cause mitochondrial dysfunction and oxidative stress, which can trigger apoptosis [8,9]. Moreover, autophagy and polymyxin-induced tubular apoptosis involving the activation of death receptors, mitochondrial damage and endoplasmic reticulum stress have also been observed [7][8][9].
The key structural features of the polymyxins are the cationic γ-diaminobutyric acid (Dab) residues, the hydrophobic D-Phe 6 , L-Leu 7 (polymyxin B) or D-Leu 6 L-Leu 7 (colistin) residues of the cyclic heptapeptide ring and the hydrophobic N-terminal fatty acyl group linked to the ring by a linear tripeptide side chain [10,11]. The antibacterial activity and toxicity of polymyxins are closely related to their structure [11]. Polymyxin analogues have previously been synthesized in an attempt to obtain high antimicrobial activity and low toxicity [12]. A group of new analogues were designed in our laboratory based on the polymyxin scaffold, with the aim of disconnecting therapeutic efficacy from toxicity [13]. To further investigate the impact of structural modifications on polymyxin nephrotoxicity, transcriptomics was employed to examine the effects of polymyxin B and two new analogues developed in our laboratory (FADDI-251 and FADDI-287) on human kidney proximal tubular HK-2 cells [14].

Networks Perturbed by Polymyxin B and Analogues
The networks perturbed by all three treatments (polymyxin B, FADDI-251 and FADDI-287) were retrieved from the SIGnaling Network Open Resource (SIGNOR) database [17] of causal relationships between biological entities ( Figure 5A). There were nine genes significantly dysregulated by all the three treatments (Table S1 (Supplementary Materials)). IKBKB (degree = 31) and IRS1 (degree = 30) were the top two hub genes by degree. IKBKB and its neighbors in the protein-protein interaction (PPI) network (e.g., FOXO, RUNX3, CASP3, MAP3K7 and MAP3K14) play essential roles in immune responses, autophagy, the cell cycle, oxidative stress-induced senescence, gene expression, protein metabolism, apoptosis and signal transduction.  Nine DEGs were induced only by polymyxin B with a degree greater than 10 in the PPI network, showing the exclusive alterations caused by polymyxin B. JUN (degree = 51) and its neighbors are involved in interleukin signaling in the immune system and RNA polymerase II transcription. STAT1 (degree = 38) and its neighbors are involved with cytokine signaling, including interferon and interleukin signaling. EGR1 (degree = 28), BDKRB2 (degree = 15) and FGFR2 (degree = 13) play essential roles in signal transduction, whereas MAP2K6 (degree = 14) and SH3RF1 (degree = 11) are associated with the immune system. The remaining two polymyxin B-specific hub genes were CEBPA (degree = 28), a transcription factor for genes regulating the cell cycle and CREB5 (degree = 12), which encodes a homodimer binding to the cAMP response element. There were no hub genes with a degree greater than 10 induced by FADDI-251 or FADDI-287. Collectively, all three polymyxins affected cellular immune responses, the cell cycle, apop-tosis and gene expression, although polymyxin B induced substantially more DEGs in these pathways.

Discussion
Nephrotoxicity is the major dose-limiting factor associated with intravenously administered polymyxins, occurring in up to 60% of patients [4]. New polymyxin analogues have been developed with the hope of increasing antibacterial activity whilst reducing nephrotoxicity [18]. Pharmacokinetic and in vitro studies have demonstrated the extraordinary accumulation of polymyxin B and colistin in the kidneys, in particular kidney tubular cells [19,20]. In this chemical biology study, we conducted transcriptomics to compare the cellular responses of human kidney proximal tubular HK-2 cells to polymyxin B and two analogues, FADDI-251 and FADDI-287. Compared to polymyxin B, the structural differences in analogues FADDI-251 and FADDI-287 involve changes at positions three (where L-Dap replaces L-Dab with one-carbon less in the side chain), six (where D-Leu replaces D-Phe) and seven (where L-Thr in FADDI-251 and L-Abu in FADDI-287 replace L-Leu). The only difference between the structures of FADDI-251 and FADDI-287 is a single hydroxyl group at position seven. These modifications maintain the key structural features of the polymyxin core but do decrease the overall hydrophobicity of the polymyxin core structure. Meanwhile, these modifications do help attenuate nephrotoxicity [14] and are critical for the interactions of polymyxins with the kidney cell membrane [21]; thus, these less nephrotoxic polymyxin analogues, FADDI-251 and FADDI-287, serve as useful tools for exploring the transcriptomic impact of the polymyxins on kidney cells to further our understanding of the mechanism of polymyxin induced-nephrotoxicity. Importantly, improved antibacterial activities of FADDI-251 and FADDI-287 have been reported against Gram-negative bacterial isolates [13]. Our current study demonstrated that, by rationally modifying multiple non-conserved positions in the polymyxin scaffold, the nephrotoxicity of new polymyxin molecules such as FADDI-287 can be significantly decreased. Importantly, a recent mouse nephrotoxicity study confirmed the reduced nephrotoxicity of FADDI-287 compared to polymyxin B in vivo [13].
According to the previously reported in vitro studies in HK-2 cells (EC 50 approximately 0.35 mM), 0.1 mM polymyxin B was employed to avoid substantial cell death and to maintain the quality of mRNA collected for subsequent transcriptomics analysis [9,22]. Cell death caused by the two polymyxin analogues was significantly lower than that with polymyxin B. A major finding of our study is that metallothionein genes and the TLR cascades were significantly perturbed by all the three polymyxins, indicating that metal homeostasis and the immune system were essential host responses to polymyxin treatment. Furthermore, vital regulators involved with immune responses, autophagy, the cell cycle, oxidative stress-induced senescence, gene expression, protein metabolism, apoptosis and signal transduction were also perturbed by all three polymyxins. Unlike FADDI-251 and FADDI-287, polymyxin B caused the largest number of DEGs, including those involved in FGFR2 and MAPK signaling transduction. Our results confirmed that hydrophobicity of position six of the polymyxin core structure is critical in the nephrotoxicity.
Metallothioneins are a family of small, cysteine-rich heavy metal-binding proteins involved in essential trace element homeostasis and metal detoxification [23]. With metalbinding and redox capabilities, metallothioneins can protect cells against metal toxicity and oxidative stress [24,25]. All three treatments resulted in the transcriptional upregulation of a large number of genes from the metallothionein family (MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1M, MT1X and pseudogene MT1L), suggesting that each induced oxidative stress. Our group and collaborators have previously reported that oxidative stress is a key factor in colistin-induced nephrotoxicity [7,26]. Dai et al. [7] reported that an oxidative stress marker, malondialdehyde, was significantly increased, and two key antioxidant enzymes (superoxide dismutase (SOD) and catalase) were significantly decreased in mouse kidneys following 7 days of colistin treatment (7.5 and 15 mg/kg/day). These results indicated a decreased ability of kidney HK-2 cells to catalyze the decomposition of reactive oxygen species. In the present transcriptomics study, we identified the metallothionein-encoding genes as potential indicators of polymyxin-induced oxidative stress.
There were seven inconsistent DEGs induced by polymyxin B and the two analogues, of which SLC38A2 and CXXC4 sparked our interest. SLC38A2 encodes the sodiumdependent neutral amino acid transporter 2 (SNAT2), and it is upregulated by amino acid starvation and hypertonicity [27]. SLC38A2 was significantly upregulated by polymyxin B but downregulated by FADDI-251 or FADDI-287, suggesting that polymyxin B caused nutrient stress via amino acid starvation in HK-2 cells. The CXXC4 protein is a negative regulator of Wnt and Ras/MAPK signaling, with the downregulation of CXXC4 promoting malignant phenotype in renal cell carcinoma by activating Wnt signaling [28,29]. As a tumor suppressor, CXXC4 inhibits cell growth by activating apoptosis in gastric cancer [30]. Interestingly, CXXC4 was downregulated by polymyxin B but upregulated by FADDI-251 or FADDI-287, indicating that apoptosis could be induced by polymyxin B. Furthermore, on the one hand, CXXC4 could be used as an indicator for polymyxin toxicity, while on the other hand, the less toxic polymyxin analogues could be considered anti-tumor drugs by targeting CXXC4.
Another major finding of our study is that polymyxin B exclusively perturbed the signaling pathways FGFR2 and MAPK, suggesting multiple signaling transduction pathways contribute to polymyxin-induced nephrotoxicity. The fibroblast growth factor (FGF) family activates FGF receptors (FGFRs) to induce pleiotropic responses that control cell proliferation, differentiation, migration, survival and shape in a context-dependent manner [31]. As receptor tyrosine kinases, FGFR1-FGFR4 are composed of an extracellular immunoglobulin-like domain, a single transmembrane domain and a cytoplasmic domain containing the catalytic protein tyrosine kinase core and regulatory sequences [32,33]. Among the FGFRs, FGFR2, which transduces FGF signals to RAS-MAPK and PI3K-AKT signaling cascades [31,34], was significantly decreased by polymyxin B. MAPK and PI3K-the AKT pathways-are fundamental signal transduction and regulation pathways involved in the majority of cellular physiological processes. Activation of these pathways is responsible for cell proliferation, differentiation, metabolism, cytoskeleton reorganization, death and survival [35][36][37][38]. Both MAPK and PI3K-AKT pathways have previously been shown to be involved in colistin-induced nephrotoxicity [7] and neurotoxicity [39], suggesting that these pathways and their downstream cell death are hallmarks of polymyxin-induced toxicity. In the present study, genes from the MAPK and PI3K-AKT pathways were only dysregulated by polymyxin B, indicating that its greater toxicity could be attributed to activation of the intracellular signaling cascades. Furthermore, the potential for reduced toxicity with FADDI-251 (hub DEGs = 14) and FADDI-287 (hub DEGs = 7) was also shown by the PPI networks where polymyxin B (hub DEGs = 26) perturbed more differentially expressed hub genes than the two analogues, including JUN, which is involved in the MAPK signaling cascade. Nine hub genes shared by all three polymyxins play crucial regulatory roles in immune responses, cell cycle, cell death, oxidative stress and signal transduction, indicating the possibility that polymyxin B and analogues all caused substantial disturbances in these functions [40]. All these responses could be associated with the physical binding of DNA and polymyxins, which directly leads to DNA damage and oxidative stress [40] and subsequent biological events, including immune responses and cell cycle arrest.

Assessment of Cell Viability
Human kidney proximal tubule HK-2 cells (ATCC CRL-2190™) were initially grown in keratinocyte serum-free medium (KSFM) supplemented with pituitary growth hormone (Life Technologies, Thermo Fisher Scientific, Scoresby, VIC, Australia) at 37 • C in a humidified atmosphere containing 5% CO 2 . For assessment of cell viability, cells were seeded and grown for 24 h in 24-well plates (0.5 × 10 5 cells/well, 1.0 mL) before treatment with

Extraction of mRNA
Cells were seeded and grown for 24 h in 6-well plates (2.5 × 10 5 cells/well, 3.0 mL) using the same growth media described above for cell viability assessments. Following treatment with 0.1 mM polymyxin B or analogues (or no treatment for controls), the growth medium was aspirated completely, and lysis buffer added to the cell-culture dish. A rubber policeman was used to detach the cells. The lysate was passed through a blunt 20-gauge needle fitted to an RNase-free syringe at least 5 times before processing according to the RNeasy Plus Mini Kit (QIAGEN, Clayton, VIC, Australia).
Quantification of the extracted mRNA was performed by the Monash Health Translation Precinct (MHTP) Medical Genomic Facility with Agilent microarray using the Human Gene Expression v2. In brief, Cyanine-3 (Cy3) labelled cRNA was prepared using the One-Color Low input Quick Amp labelling Kit (Agilent, Mulgrave, VIC, Australia), followed by RNeasy column purification as described above. Dye incorporation and cRNA yield were checked with a NanoDrop ND-1000 Spectrophotometer. The Cy3 labeled cRNA was fragmented and hybridized with the Human Gene Expression v2 at 65 • C for 17 h. After washing, the slides were scanned with a DNA microarray scanner using one-color scan settings for 8x60k array slides (GeneSpring, GX11.5; Agilent). The scanned images were analyzed with Feature Extraction Software 11.0.1.1 (Agilent) using default parameters.

Bioinformatic Analysis
The raw intensities were pre-processed to perform background correction, quantile normalization and log 2 transformation by Bioconductor package Limma [42]. If multiple probes were mapped to the same gene, the expression value for the gene was summarized as the arithmetic mean of the values of the multiple probes (on the log 2 scale). DEGs were identified using analysis of variance (ANOVA) with an FDR < 0.2. All p-values in this paper were adjusted by the Benjamini-Hochberg FDR procedure [43]. The SIGNOR database [17], which has collected approximately 12,000 causal relationships between over 2800 human proteins, was employed to construct the directed gene network. Networks were visualized by Cytoscape [44]. The functional pathway analysis was performed against Reactome hierarchy [16]. The hypergeometric distribution was used to determine whether a pathway was over-represented in the interesting gene list (i.e., DEG list).

Conclusions
This study revealed metal homeostasis by metallothioneins and Toll-like receptor cascades as major cellular responses involved in polymyxin-induced toxicity. Only polymyxin B activated the FGFR2 and MAPK signaling pathways, indicating that decreasing the